Benzoxazole compounds and methods of use

ABSTRACT

The present invention provides benzoxazole compounds and pharmaceutically acceptable salts thereof and pharmaceutical compositions including the same. The present invention further provides methods of use as described herein.

BACKGROUND OF THE INVENTION

Many early immune responses to damage or infection are initiated by afamily of innate immune toll-like receptors (TLRs). The TLR family hasbroad specificity, recognizing molecular structures that are highlyconserved among pathogenic organisms or different types of physicaldamage. The ligands of specific TLRs include endotoxin, single- ordouble-stranded RNA, peptidoglycan, flagellin, heat shock proteins, andin the case of TLR9, unmethylated CpG sequences in DNA (Bell et al.,Trends Immunol. 24:528-533 (2003) and Wagner, Trends Immunol. 25:381-6(2004)). While this early sensing of disease or damage is critical tothe ensuing immune response, it can also be the source of inappropriateor damaging responses as well. For example, TLR9 has been implicated inautoimmune disorders, including lupus, which has long been associatedwith anti-DNA antibody reactivity, and multiple sclerosis (Prinz et al.,J Clin Invest 116:456-464 (2006)). TLR9 also appears to underlie theuncontrolled inflammatory response that is associated with death fromseptic shock (Plitas et al., J Exp Med 205: 1277-83 (2008)).

TLR9 was first identified as an innate immune receptor expressed byhuman B cells and plasmacytoid dendritic cells (PDC) that bindsunmethylated CpG sequences, resulting, in cellular activation andcytokine secretion (Hemmi et al., Nature 408:740-745 (2000)). Thesesequences are over-represented in bacterial DNA in comparison toeukaryotic DNA, and therefore can serve as an indicator of bacterialinfection. However, eukaryotic DNA contains some unmethylated CpG and iscapable of stimulating TLR9 as well (Vallin et al., J Immunol.163:6306-13 (1999) and Leadbetter et al. (2002)). While DNA from dyingcells is typically ingested and sequestered from the system, lupus isassociated with genetic defects that lead to poor clearance andaccumulation of excessive cell debris (Krislman et al., Seminars inImmunology 18:240-243 (2006)), exposing, the immune system to abnormallyhigh levels of ligand. This condition, combined with targeted uptake ofDNA-containing complexes by either complex-specific immunoglobulin on Bcells, or Fc receptors on dendritic or antigen-presenting cells, canresult in presentation and reaction to autoantigen, a response boostedby the presence of TLR9 ligand in the stimulating complex. Thisobservation is exemplified by the fact that PDC respond to complexesfound in systemic lupus erythematosus (SLE) serum by secretingα-interferon, and that this is Fc receptor and DNA-dependent andmediated by TLR9 (Leadbetter et al., (2002) and Means et al., J ClinInvest. 1152:407-17 (2005)). The resulting α-interferon can furtherdrive dendritic cell and B cell maturation. Correspondingly, array datashow activation of α-interferon inducible genes in patients with severedisease (Bennett et al., J Exp Med. 197:711-23 (2003)). As PDC are thepredominant source of α-interferon in the body, this observation furtherassociates this cell population with disease.

B cells are also central to lupus autoimmunity, and are driven toproliferate and produce IL-6 and antibodies by TLR9 stimulation withsynthetic oligonucleotides. Due to preferential association with B cellsexpressing autoreactive cell surface immunoglobulin,autoantigen-specific cells will predominate in this response. Aself-reinforcing cycle is thus at work, in which DNA or RNA-containingcell debris combines with antibodies from TLR9-stimulatedautoantigen-specific B cells to stimulate plasmacytoid dendritic cellsvia Fc receptors.

SUMMARY OF THE INVENTION

A first embodiment of the present invention provides a compound offormula (I):

or a pharmaceutically acceptable salt thereof,

wherein one of R⁵, R⁶, or R⁷ is a group of formula (a):

and when R⁵ is (a), R⁶ and R⁷ are both H, when R⁶ is (a), R⁵ and R⁷ areboth H, and when R⁷ is (a), R⁵ and R⁶ are both H;

i and j are the same and are 0, 1, 2, 3, or 4;

R¹ and R⁴ are the same and selected from the group consisting of H, CH₃,and CH₂CH₃;

R² is CH₃ and R³ is selected from the group consisting of(CH₂)_(h)N(CH₃)₂ and (CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(CH₃)₂, wherein h is 2, 3,or 4; or

R² and R³ are the same and selected from the group consisting of:

-   -   (CH₂)_(k)CH₃, wherein k is 0, 1 or 2;    -   (CH₂)_(m)N(CH₂CH₃)₂, wherein m is 2 or 3;    -   (CH₂)_(n)N(CH₃)₂, wherein n is 2, 3 or 4;    -   (CH₂)_(p)—O—(CH₂)_(g)N(CH₃)₂, wherein p and q are the same and        are 2 or 3;    -   a group of formula (b):

-   -    wherein u is 0 or 1; or    -   a group of formula (c):

-   -    wherein v is 0 or 1, Z is N or CH,    -   and X is O or NCH₃; or

R¹—N—R² and R³—N—R⁴ are the same and selected from the group consistingof:

-   -   a group of formula (d):

-   -    and    -   a group of formula (e):

-   -   -   wherein            -   r is 1, 2, or 3,            -   Y is CH or N,            -   R⁸ is H, CH₃, CH(CH₃)₂, N(CH₃)₂, CH₂OCH₃, or a group of                formula (f):

-   -   -   -    wherein t is 0 or 1, and            -   R⁹ is H, CH₂OCH₃, or a group of formula (f).

A second embodiment of the present invention provides a pharmaceuticalcomposition including a compound as described herein.

A third embodiment of the present invention provides a method oftreating an immunological disorder such as sepsis, lupus, rheumatoidarthritis, or multiple sclerosis in a subject, including administeringto the subject a compound as described herein in an effective amount.

A fourth embodiment of the present invention provides a method oftreating lupus in a subject, including administering to the subject acompound as described herein in an effective amount.

A fifth embodiment of the present invention provides the use of acompound as described herein for the manufacture of a medicament fortreating immunological disorders such as sepsis, lupus, rheumatoidarthritis, or multiple sclerosis.

Other embodiments of the present invention are disclosed herein anddiscussed in greater detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing the concentration of interleukin 6 (IL-6)detected in lymph nodes removed from mice after two weeks ofadministration of 20 mg/kg or 60 mg/kg of Compound 2, Compound 23, orhydroxychloroquine (HCQ) in vivo, and stimulation of the lymph nodes exvivo with oligo CpG 1668, as described in Example 11.

FIG. 2 is a plot showing the results of optical density measurements at450 nanometers (OD₄₅₀) in an ELISA assay of mouse anti-dsDNA, before andafter 7 weeks of dosing of MRL/MpJ-faslpr/J (“MRL/lpr”) mice with 20mg/kg or 60 mg/kg of Compound 23, as described in Example 12A.

FIG. 3 is a set of bar graphs showing the results of anti-nuclearantibodies (ANA) testing in the MRL/lpr spontaneous lupus model of miceadministered 20 mg/kg or 60 mg/kg of Compound 23 after 4, 10 or 12 weeksof dosing, as described in Example 12A. The results are shown as thenumber of mice scored in each of five possible categories of ANA results(−, −/+, 0, +, or ++).

FIG. 4 is a graph showing results of an ELISA assay at OD₄₅₀ of MRL/lprmouse anti-dsDNA pre- and post-dosing for 12 weeks with Compound 23 (20mg/kg or 60 mg/kg), HCQ, or cyclophosphamide, as described in Example12B.

FIG. 5 is a plot showing results of an ELISA assay at OD₄₅₀ of mouseanti-dsDNA pre- and post-dosing of NZB/WF1/J mice for 16 weeks with 20mg/kg or 60 mg/kg Compound 23 or Compound 30, as described in Example13.

FIG. 6 is a set of bar graphs showing the number of NZB/WF1/J mice witheach of the five possible ANA scores, after 16 weeks of dosing with 20mg/kg or 60 mg/kg of Compound 23 or Compound 30, as described in Example13

FIG. 7 is a graph showing results of administration of 20 mg/kg or 60mg/kg of Compound 23 to C57BL/6J mice immunized with synthetic peptideMOGp35-55 to induce experimental autoimmune encephalitis (EAE), whichmimics multiple sclerosis, as described in Example 14.

FIG. 8 is a graph showing results of administration of 6 mg/kg, 20 mg/kgor 60 mg/kg Compound 2 to male DBA/1J mice immunized with bovine type IIcollagen emulsified in complete Freund's adjuvant to induce arthritis,as described in Example 15.

DETAILED DESCRIPTION A. Definitions

“Treatment”, “treat”, and “treating” refer to reversing, alleviating, orinhibiting the progress of a disorder or disease as described herein.Moreover, as used herein, “treatment” of a subject includes theapplication or administration of a compound of the invention describedherein to a subject, or application or administration of the compound toa cell or tissue from a subject, who has an immunological disorder, hasa symptom of such an immunological disorder, or is at risk of (orsusceptible to) such an immunological disorder, with the purpose ofcuring, healing, alleviating, relieving, altering, remedying,ameliorating, improving, or affecting the disorder, the symptom of thedisorder, or the risk of (or susceptibility to) the disorder. The term“treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the subject; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating;improving a subject's physical or mental well-being; or, in somesituations, preventing the onset of a more severe form or symptom of thedisorder. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including, the results of a physicalexamination, a psychiatric evaluation, or a diagnostic test known in theart.

In one embodiment, the term “treating” includes improving a symptom ofan immunological disorder such as improvement or relief frominflammation, infection, fever, altered mental status, organdysfunction, anemia, joint pain, fatigue, cognitive difficulties,spasticity and/or tremors caused by or associated with an immunologicaldisorder.

“Prevention”, “prevent”, and “preventing” refer to eliminating orreducing the incidence or onset of a disorder or disease as describedherein, as compared to that which would occur in the absence of themeasure taken.

“Effective amount” refers to an amount that causes relief of symptoms ofa disorder or disease as noted through clinical testing and evaluation,patient observation, and/or the like. An “effective amount” can furtherdesignate a dose that causes a detectable change in biological orchemical activity. The detectable changes may be detected and/or furtherquantified by one skilled in the art for the relevant mechanism orprocess. Moreover, an “effective amount” can designate an amount thatmaintains a desired physiological state, i.e., reduces or preventssignificant decline and/or promotes improvement in the condition ofinterest. An “effective amount” can further refer to a therapeuticallyeffective amount.

“Subject”, as used herein, means a mammalian subject (e.g., dog, cat,horse, cow, sheep, goat, monkey, etc.), and particularly human subjects(including both male and female subjects, and including neonatal,infant, juvenile, adolescent, adult and geriatric subjects, and furtherincluding various races and ethnicities including, but not limited to,white, black, Asian, American Indian and Hispanic).

As used herein, the term “a pharmaceutically acceptable salt” refers tothe relatively non-toxic, inorganic and organic acid salts of compoundsof the invention. These salts can be prepared in situ during, the finalisolation and purification of the compounds or by reacting the purifiedcompound in its free form separately with a suitable organic orinorganic acid and isolating the salt thus formed. Representative acidsalts include acetate, adipate, aspartate, benzoate, besylate,bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate,cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate,glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinafoate salts. In one embodiment, thepharmaceutically acceptable salt is a hydrochloride/chloride salt.

B. Compounds

Some of the compounds described herein can comprise one or moreasymmetric centers, and thus, the compounds can exist in variousisomeric forms, e.g., stereoisomers and/or diastereomers. When theorientation of a bond around a chiral center is not specified in aformula, it is to be understood that the formula encompasses everypossible isomer such as geometric isomer, optical isomer, stereoisomerand tautomer based on asymmetric carbon, which can occur in thestructures of the inventive compounds.

As noted above, the present invention provides a compound of formula(I):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R⁵, R⁶,and R⁷ are defined as described herein above.

In some embodiments of the compound of formula (I), i and j are both 1.

In further embodiments, R² and R³ are the same and selected from thegroup consisting of:

-   -   (CH₂)_(k)CH₃, wherein k is 0, 1 or 2;    -   (CH₂)_(m)N(CH₂CH₃)₂, wherein m is 2 or 3;    -   the group of formula (b) described herein above; and    -   the group of formula (c) described herein above.

According to other embodiments, R¹—N—R² and R³—N—R⁴ are the same and areselected from:

-   -   a group of formula (g): a group of formula (h):

-   -   a group of formula (i): a group of formula (j):

-   -   a group of formula (k): a group of formula (m):

In some embodiments of the compound of formula (I):

i and j are both 1;

R¹ and R⁴ are the same and selected from the group consisting of H andCH₃;

R² is CH₃ and R³ is selected from the group consisting of (CH₂)₂N(CH₃)₂and (CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(CH₃)₂, or

R² and R³ are the same and selected from the group consisting of:

-   -   a) (CH₂)_(k)CH₃, wherein k is 0, 1, or 2;    -   b) (CH₂)_(m)N(CH₂CH₃)₂, wherein M is 2 or 3;    -   c) the group of formula (b) described herein above; and    -   d) the group of formula (c) described hereinabove; or

R¹—N—R² and R³—N—R⁴ are the same and selected from the group consistingof:

-   -   the group of formula (g), the group of formula (h), the group of        formula (i),    -   the group of formula (j), the group of formula (k), and the        group of formula    -   (m) all as described herein above.

In further embodiments, R⁵ is the group of formula (a) describedhereinabove, and R⁶ and R⁷ are each H.

In particular embodiments of the compound of formula (I), the compoundhas a structure selected from:

Compound Structure Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Compound 7

Compound 8

Compound 9

Compound 10

Compound 11

Compound 12

Compound 13

Compound 14

Compound 15

Compound 16

Compound 17

Compound 18

Compound 19

Compound 20

Compound 21

Compound 22

Compound 23

Compound 24

Compound 25

Compound 26

Compound 27

Compound 28

Compound 29

Compound 30

Compound 31

Compound 32

Compound 33

Compound 34

Compound 35

Compound 36

Compound 37

Compound 38

In another embodiment, the compound of Formula I is selected from thegroup consisting of Compound 1, Compound 2, Compound 10, Compound 11,Compound 12, Compound 13, Compound 14, Compound 16, Compound 17,Compound 26, Compound 29, Compound 30, Compound 31, Compound 32,Compound 34, and Compound 35 or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is selected from the groupconsisting of Compound 13, Compound 16, Compound 22, Compound 23,Compound 26, Compound 27, Compound 30, Compound 34, and Compound 35 or apharmaceutically acceptable salt thereof. In another embodiment, thecompound is selected from the group consisting of Compound 23, Compound26, and Compound 30, or a pharmaceutically acceptable salt thereof. Inyet another embodiment, the compound is Compound 23 or apharmaceutically acceptable salt thereof.

C. Synthesis of Compounds

Compounds described herein can be prepared by a sequence of reactions.General methods for preparing compounds of the present invention are setforth below. In certain cases, a particular compound is described by wayof example as presented further below in the section describing theexamples. Except as provided below, in the following methods, R¹, R²,R³, R⁴, i, and j are the same as defined in Formula I, above.

Method 1—Alkylation, Amination or Alkylation, Amination andN-Methylation

In the reaction scheme of Method 1 shown above:

-   -   n, i, and j are the same and are 0, 1, 2, 3, or 4;    -   R^(1a) and R^(4a) are both the same as R^(x);    -   R^(2a) and R^(3a) are both the same as R^(y);    -   R^(1b) and R^(4b) are the same;    -   R^(2b) and R^(3b) are the same;    -   when R^(1a) and R^(4a) are both H, R^(1b) and R^(4b) are both        methyl; and    -   when R^(2a) and R^(3a) are both H, R^(2b) and R^(3b) are both        methyl.

In one method of synthesizing symmetrical compounds according toembodiments of the invention, 2-(4-hydroxyphenyl)benzoxazol-6-ol (1A)may be reacted with a bromochloroalkane (1B), e.g., in the presence ofpotassium carbonate in dimethyl sulfoxide (DMSO), to form thedichloroether derivative (1C). 2-(3-hydroxyphenyl)benzoxazol-6-ol and2-(2-hydroxyphenyl)benzoxazol-6-ol may also be used in lieu of compound1A. The dichloroether derivative (1C) may then be reacted with an amine(HNR^(x)R^(y)) to form diamine (1D). In the synthetic scheme for Method1, the amine is generically referred to as HNR^(x)R^(y) to indicate thata single amine reactant may be used to form the amine groups—NR^(1a)R^(2a) and —NR^(3a)R^(4a) in diamine (1D). As such, R^(x) is thesame as R^(1a) and R^(4a), and R^(y) is the same as R^(2a) and R^(3a).Additionally, if R^(1a) and R^(4a) are hydrogen, or if R^(2a) and R^(3a)are hydrogen, the compound may optionally undergo N-methylation to formthe N-methyl derivatives (1E). Compounds 1D and 1E may both be compoundsaccording to embodiments of the invention.

Method 2—Mitsunobu Reaction or Mitsunobu Reaction and N-Methylation

In the reaction scheme of Method 2 shown above:

-   -   n, i, and j are the same and are 0, 1, 2, 3, or 4;    -   R^(1a) and R^(4a) are both the same as R^(x):    -   R^(2a) and R^(3a) are both the same as R^(y); and    -   R^(1b) and R^(4b) are the same;    -   R^(2b) and R^(3b) are the same;    -   when R^(1a) and R^(4a) are both H, R^(1b) and R^(4b) are both        methyl; and    -   when R^(2a) and R^(3a) are both H, R^(2b) and R^(3b) are both        methyl.

In another method for synthesizing symmetrical compounds according toembodiments of the invention, 2-(4-hydroxyphenyl)benzoxazol-6-ol (2A)may be reacted with a hydroxyalkylamine (2B), via the Mitsunobureaction, to produce a diamine (2C). 2-(3-hydroxyphenyl)benzoxazol-6-oland 2-(2-hydroxyphenyl)benzoxazol-6-ol may also be used in lieu ofcompound 2A. Additionally, if R^(1a) and R^(4a) are hydrogen, or ifR^(2a) and R^(3a) are hydrogen, the compound may optionally undergoN-methylation to form the N-methyl derivatives (2D). Compounds 2C and 2Dmay both be compounds according to embodiments of the invention.

Method 3—for Asymmetric Side Chain Analogs

In the reaction scheme of Method 3:

-   -   R¹ and R⁴ are the same and selected from the group consisting of        H and CH₃; and    -   R² is CH₃ and R³ is selected from the group consisting of        (CH₂)₂N(CH₃)₂, and (CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(CH₃)₂.

Asymmetric compounds according to some embodiments of the invention maybe synthesized via Method 3. In such case, 4-aminobenzene-1,3-diol (3A)may be reacted with a benzoic acid having a protected hydroxyl group,such as 4-(benzyloxy)benzoic acid (3B), 3-(benzyloxy)benzoic acid or2-(benzyloxy)benzoic acid in the presence ofbenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (PyBop), to form the benzamide derivative (3C). Thebenzamide may then cyclized to form the benzoxazole ring (3D). At thispoint, either Method 1 or Method 2, described above, may be used toconvert the hydroxy-functional benzoxazole (3D) to the amine (3E).Referring to Methods 1 and 2, R¹ may be R^(1a) or R^(1b) and R² may beR^(2a) or R^(2b). The benzyl ether group may then be deprotected, e.g.,by hydrogenation with palladium, to form the hydroxy-functional amine(3F). Then, either Method 1 or Method 2 may be used to transform thehydroxyl-functional amine to the diamine (3G). Referring to Methods 1and 2, R⁴ may be R^(4a) or R^(4b) and R³ may be R^(3a) or R^(3b).

D. Pharmaceutical Compositions

In one embodiment, the present invention is a pharmaceutical compositioncomprising the compound. In another embodiment, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable carrier” as used herein refers to anysubstance, not itself a therapeutic agent, used as a vehicle fordelivery of a therapeutic agent to a subject.

The compositions of the present invention may be suitable forformulation for oral, parenteral, inhalation spray, topical, rectal,nasal, sublingual, buccal, vaginal or implanted reservoiradministration, etc. Preferably, the compositions are administeredorally, topically, intraperitoneally or intravenously. Sterileinjectable forms of the compositions of this invention may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a nontoxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

A pharmaceutically acceptable oil may be employed as a solvent orsuspending medium in compositions of the present invention. Fatty acids,such as oleic acid and its glyceride derivatives are suitably includedin injectable formulations, as are natural pharmaceutically acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. The oil containing compositions of thepresent invention may also contain a long-chain alcohol diluent ordispersant, such as carboxymethyl cellulose or similar dispersing agentsthat are commonly used in the formulation of pharmaceutically acceptabledosage forms including emulsions and suspensions. The compositionssuitably further comprise surfactants (such as non-ionic detergentsincluding Tween® or Span®) other emulsifying agents, or bioavailabilityenhancers.

The compositions of this invention may be in the form of an orallyacceptable dosage form including, but not limited to, capsules, tablets,suspensions or solutions. The oral dosage form can include at least oneexcipient. Excipients used in oral formulations of the present caninclude diluents, substances added to mask or counteract a disagreeabletaste or odor, flavors, dyes, fragrances, and substances added toimprove the appearance of the composition. Some oral dosage forms of thepresent invention suitably include excipients, such as disintegrants,binding agents, adhesives, wetting agents, polymers, lubricants, orglidants that permit or facilitate formation of a dose unit of thecomposition into a discrete article such as a capsule or tablet suitablefor oral administration additional. Excipient-containing tabletcompositions of the invention can be prepared by any suitable method ofpharmacy which includes the step of bringing into association one ormore excipients with a compound of the present invention in acombination of dissolved, suspended, nanoparticulate, microparticulateor controlled-release, slow-release, programmed-release, timed-release,pulse-release, sustained-release or extended-release forms thereof.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be in the form of a suppository for rectal administration.The suppositories can be prepared by mixing the agent with a suitablenon-irritating excipient that is solid at room temperature but liquid atrectal temperature and therefore will melt in the rectum to release thedrug. Such materials include cocoa butter, beeswax and polyethyleneglycols.

Pharmaceutically acceptable compositions of the present invention may bein the form of a topical solution, ointment, or cream in which theactive component is suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Where the topical formulation is inthe form of an ointment or cream, suitable carriers include, but are notlimited to, mineral oil, sorbitan monostearate, polysorbate 60, cetylesters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol andwater.

When the pharmaceutically acceptable composition is an ophthalmicformulation, it may be a micronized suspension in isotonic, pH adjustedsterile aqueous solution, or as a solution in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in the formof an ointment.

The pharmaceutically acceptable compositions of this invention may alsobe administered by nasal, aerosol or by inhalation administrationroutes. Such compositions are prepared according to techniqueswell-known in the art of pharmaceutical formulation and may be preparedas solutions in saline, employing, benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

Additionally, the pharmaceutical formulation including compounds of thepresent invention can be in the form of a parenteral formulation.

In certain embodiments, the pharmaceutically acceptable compositions ofthis invention are formulated preferably for oral administration.

E. Methods of Use

In another embodiment, the present invention is a method of preventingor treating an immunological disorder in a subject comprisingadministering a subject a therapeutically effective amount of a compoundof the invention. In a particular embodiment, the compound of thepresent invention used to prevent or treat an immunological disorder,according to methods of the present invention, is a compound of FormulaI selected from the group consisting of: Compound 13, Compound 16,Compound 22, Compound 23, Compound 26, Compound 27, Compound 30,Compound 34, and Compound 35 or a pharmaceutically acceptable saltthereof. In another embodiment, the compound is selected from the groupconsisting of Compound 23, Compound 26, and Compound 30 or apharmaceutically acceptable salt thereof. In another embodiment, thecompound is Compound 23 or a pharmaceutically acceptable salt thereof.

In one embodiment the immunological disorder prevented or treated withthe method of the present invention is an autoimmune disease. As usedherein the term “autoimmune disease” refers to maladies that aregenerally caused by the failure of the immune system to distinguish selfcomponents from non-self components. Autoimmune diseases that can beprevented or treated by compounds of the present invention include, butare not limited to, lupus (such as SLE), pemphigus vulgaris, myastheniagravis, hemolytic anemia, thrombocytopenia purpura, Grave's disease,Sjogren's disease, dermatomyositis, Hashimoto's disease, polymyositis,inflammatory bowel disease, multiple sclerosis, diabetes mellitus,ulcerative colitis, rheumatoid arthritis, scleroderma and psoriasis. Inparticular embodiments of the present invention, the autoimmune diseaseis lupus or multiple sclerosis. In some embodiments, the autoimmunedisease is SLE. As used herein, lupus refers to an autoimmune diseasecharacterized by antibodies against self-cellular components includingDNA, RNA, histones, and nucleosomes. Physical symptoms include kidneydisease resulting, from autoantibody or immune complex deposition in theglomeruli, cutaneous rash, photosensitivity, arthritis and neurologicdisorders including seizures and psychosis. The disease predominantlyaffects women, and usually strikes young, adults. While rarely directlyfatal, it is associated with increased mortality and has a severe impacton quality of life, with ongoing fatigue, weight loss, atheroscleroticrisk, and fever.

In another embodiment, the immunological disorder is sepsis. As usedherein, sepsis refers to a systemic inflammatory response caused by aninfection. Compounds of the present invention may be administered totreat severe sepsis and septic shock, both of which may result fromsepsis. In particular embodiments, compounds of the present inventionmay be administered to treat severe sepsis. In other embodiments,compounds of the present invention may be used to treat septic shock.

According to further embodiments of the present invention, the compoundsdescribed herein can used for the treatment of lupus, multiple sclerosisand/or rheumatoid arthritis during relatively short periods of time whenthe disease flares such that symptoms of the disease increase inintensity and/or type. In some embodiments, the compounds describedherein can be used as a chronic treatment, e.g., treatment that lastsdays, months or years.

In some embodiments of the present invention, the compounds describedherein can be co-administered with an additional agent useful fortreating lupus, i.e., a combination therapy. The additional agent usefulfor treating lupus can be one or more of the following classes of drugs:steroids (e.g., glucocorticoids and corticosteroids),immunosuppressives, nonsteroidal anti-inflammatory drugs (NSAIDs),antibody therapies, interferons, cytotoxics and antimalarials. In someembodiments, the additional agent useful for treating lupus is at leastone of the following: dehydroepiandrosterone (DHEA), prednisone,prednisolone, methylprednisolone, hydrocortisone, fludrocortisone,clobetasol, halobetasol, triamcinolone, betamethasone, fluocinolone,fluocinonide, leflunomide, azathioprine, methotrexate, mitoxantrone,cladribine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate(including mycophenoloc acid, mycophenolate mofetil and mycophenolatesodium), celecoxib, diclofenac, fenoprofen, flurbiprofen, ibuprofen,ketoprofen, fenbufen, indomethacin, meclofamate, meloxicam, nabumetone,naproxen, oxaprozin, rofecoxib, salicylates, acetylsalicylic acid,acetaminophen, sulindac, tolmetin, rituximab, belimumab, infliximab,adalimumab, epratuzumab, natalizumab, α-interferon and/or quinine andquinine derivatives such as hydroxychloroquine, chloroquine, quinidineand quinacrine.

In some embodiments, the compounds described herein can beco-administered with an additional agent useful for treating multiplesclerosis, i.e., a combination therapy. The additional agent useful fortreating multiple sclerosis can be one or more of the following classesof drugs: steroids (e.g., glucocorticoids and corticosteroids),immunosuppressives, antibody therapies and interferons. In someembodiments, the additional agent useful for treating multiple sclerosisis at least one of the following: dehydroepiandrosterone, prednisone,prednisolone, methylprednisolone, hydrocortisone, fludrocortisone,clobetasol, halobetasol, triamcinolone, betamethasone, fluocinolone,fluocinonide, azathioprine, methotrexate, novantrone, copaxone,cladribine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolate(including mycophenoloc acid, mycophenolate mofetil and mycophenolatesodium), rituximab, belimumab, epratuzumab, natalizumab and/orβ-interferons.

In still other embodiments, the compounds described herein can beco-administered with an additional agent useful for treating rheumatoidarthritis, i.e., a combination therapy. The additional agent useful fortreating rheumatoid arthritis can be one or more of the followingclasses of drugs: cyclooxygenase-2 (COX-2) inhibitors, NSAIDs,disease-modifying antirheumatic drugs (DMARDs), steroids,immunosuppressives and antibody therapies. In some embodiments, theadditional agent useful for treating rheumatoid arthritis is at leastone of the following: celecoxib, diclofenac, fenoprofen, flurbiprofen,ibuprofen, ketoprofen, fenbufen, indomethacin, meclofamate, meloxicam,nabumetone, naproxen, oxaprozin, rofecoxib, acetylsalicylic acid,acetaminophen, sulindac, tolmetin, dehydroepiandrosterone, prednisone,prednisolone, methylprednisolone, hydrocortisone, fludrocortisone,clobetasol, halobetasol, triamcinolone, betamethasone, fluocinolone,fluocinonide, leflunomide, azathioprine, methotrexate, mitoxantrone,cladribine, chlorambucil, cyclophosphamide, cyclosporine, mycophenolatemofetil, rituximab, belimumab, infliximab, adalimumab, epratuzumab,natalizumab, anakinra, abatacept, auranofin, aurothioglucose,aurothiomalate, sulfasalazine, minocycline and/or quinine and quininederivatives such as hydroxychloroquine, chloroquine, quinidine andquinacrine.

In some embodiments, the compounds described herein can beco-administered with an additional agent useful for treating sepsis,i.e., a combination therapy. The additional agent useful for treatingsepsis can be one or more of the following classes of drugs:antibiotics, aminoglycosides, colloid or crystalloid fluids (i.e., fluidtherapy), vasopressors, inotropic drugs, steroids and anticoagulants. Insome embodiments, the additional agent useful for treating sepsis is atleast one of the following: albumin, starch, norepinephrine, dopamine,vasopressin, dobutamine, prednisone, prednisolone, methylprednisolone,hydrocortisone, fludrocortisone, and activated protein C including, butnot limited to, human recombinant activated protein C (rhAPC).

In further embodiments, the compounds can be used to decrease the dosesof other treatments used in combination to treat the disorders describedherein, for example, in steroid sparing regimens.

In some embodiments, the compounds described herein are administeredprior to, concurrently with or subsequent to the administration of theadditional agent useful for treating lupus, multiple sclerosis,rheumatoid arthritis and/or sepsis.

Administration of the compounds described herein “prior to”administration of the additional agent refers to administering thecompounds described herein to a subject prior to initial treatment withthe additional agent, or prior to administration of the additional agentduring a treatment protocol that includes administering the compoundsdescribed herein and the additional agent to a subject population thatis at risk or afflicted with lupus, multiple sclerosis, rheumatoidarthritis and/or sepsis.

With concurrent administration, the compounds described herein and theadditional agent are administered at the same point in time orimmediately following one another. In general, the compounds describedherein and the additional agent are administered at times sufficientlyclose that the results observed are relatively indistinguishable fromthose achieved when they are administered at the same point in time.

The compounds may be administered to subjects by any suitable route,including orally (inclusive of administration via the oral cavity andfurther including administration via an orogastric feeding tube),parenterally, by inhalation spray, topically, transdermally, rectally,nasally (including a nasogastric feeding tube), sublingually, buccally,vaginally or via an implanted reservoir. The term “parenteral” as usedherein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,parenterally, transdermally or by inhalation spray.

Compounds are administered to the subjects in an effective amount. Aneffective amount is generally 0.01 mg/kg to 500 mg/kg body weight perday. In some embodiments, the pharmaceutically acceptable compositionsmay be formulated so that a dosage of between 0.01 mg/kg to 100 mg/kgbody weight per day of the compound can be administered to a patientreceiving these compositions. In certain embodiments, the compositionsof the present invention are formulated to provide a dosage of between0.01 mg and 70 mg. In other embodiments, the compositions of the presentinvention are formulated to provide a dosage of between 0.1 mg and 25 mgor between 5 mg and 60 mg.

In some embodiments, the effective dose is between about 5 and 250mg/kg, between about 10 and 200 mg/kg, or between about 20 and 120mg/kg. In some embodiments, effective dosages include 5 mg/kg, 10 mg/kg,20 mg/kg, 25 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 75 mg/kg, 100 mg/kg,120 mg/kg and 150 mg/kg. Dosage forms can be in the form, e.g., oftablets or capsules, and the effective dose may be provided in one ormore tablets, capsules or the like, and be provided once a day orthroughout the day at intervals, e.g., of 4, 8 or 12 hours. Tablets orcapsules, for example, could contain, e.g., 10, 25, 50, 75, 100, 150,200 mg of compound. Liquid formulations could also be prepared so thatany dosage could readily and conveniently be dispensed.

In some embodiments, the amount of the compounds of the presentinvention that may be combined with the excipient materials to produce acomposition in a single dosage form will vary depending, upon the hosttreated, and the particular route of administration.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, gender, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner. Acomparator compound is used in several of the examples below. Theformula of the comparator is as follows:

Exemplary compounds of the present invention were prepared in accordancewith the schemes and experimental designs described below.

Example 1 Synthesis of Compound 40

Compound 40. As depicted above, 1-bromo-3-chloropropane (7.3 mL, 0.074mol) was added to a stirring mixture of Compound 39 (5.62 g, 0.0247 mol)and potassium carbonate (10 g, 0.074 mol) in DMSO (50 mL) at roomtemperature and the resulting mixture was stirred for 4 hours andthin-layer chromatography (TLC) showed the reaction was completed. Themixture was then poured into water (200 mL), extracted three times in150 mL ethyl acetate (EtOAc), dried over Na₂SO₄, filtered andconcentrated. Vacuum chromatography (10% to 30% EtOAc/hexane) of theresidue gave a white solid product Compound 40 (8.10 g, 86%).Crystallization in hexane/EtOAc gave a white pure solid product (6.40 g)and mother liquor with minor impurities (1.60 g). ¹H NMR (400 MHz,CD₃OD) δ 8.11 (d, J=9.1 Hz, 2H), 7.55 (d, J=8.8 Hz, 1H), 7.27 (d, J=2.3Hz, 1H), 7.11 (d, J=9.1 Hz, 2H), 7.00 (dd, J=2.3, 8.8 Hz, 1H), 4.22 (t,J=5.9 Hz, 2H), 4.19 (t, J=6.15, 2H), 3.78 (tt, J=6.4 Hz, 4H), 2.30 (dt,J=1.76, 1.76 Hz, 4H). For preparation of starting compound Compound 39,see J. Med. Chem. 2004, 47, 5021-5040.

The same reaction was performed with isomers of Compound 39. IsomersCompound 47 and Compound 48 were prepared via the synthetic procedure inJ. Med. Chem. 2004, 47, 5021-5040.

Example 2 Synthesis of Compound 49

Compound 49. Replacement of 1-Bromo-3-chloropropane with1-Bromo-2-chloroethane in Example 1 gave Compound 49.

Example 3 Synthesis of Compound 50

Compound 50. Replacement of 1-Bromo-3-chloropropane with1-Bromo-5-chloropentane in Example 1 gave Compound 50.

Example 4 Synthesis of Compound 23

Compound 23. Referring to the scheme above, a mixture of Compound 40(8.40 g, 0.0221 mol) produced as described in Example 1 and pyrrolidine(7.4 mL, 0.088 mol) in ethanol (50 mL) was heated at 100° C. for 12hours under sealed tube. TLC (20% triethylamine in methanol (Et₃N/MeOH))showed a single new spot and mass spectroscopy showed one single desiredpeak of 450 (M+H). The mixture was cooled to room temperature,concentrated and the excess pyrrolidine was removed by azeotropicconcentration with toluene (three times in 50 mL). The residue wasdissolved in ethanol (EtOH), filtered and concentrated. The residualsolid was crystallized in MeOH/EtOAc to give pure product Compound 23(7.88 g, 68%). ¹H NMR (400 MHz, CD₃OD) δ 8.12 (d, J=8.9 Hz, 2H), 7.57(d, J=8.8 Hz, 1H), 7.28 (d, J=2.2 Hz, 1H), 7.12 (d, J=8.9 Hz, 2H), 7.01(dd, J=2.2, 8.8 Hz, 1H), 4.18 (t, J=5.86 Hz, 2H), 4.15 (t, J=6.15 Hz,2H), 3.07-2.94 (m, 12H), 2.15 (m, 4H), 1.96 (m, 8H); MS (ES⁺) m/z 450.4.

Example 5 Synthesis of Compound 30

Compound 30. Referring to the scheme above, a mixture of Compound 40(13.4 g, 0.0352 mol) produced as described in Example 1 and piperidine(14 mL, 0.14 mol) in ethanol (100 mL, 2 mol) was heated at 100° C. for18 hours under sealed tube. TLC (10% Et₃N/MeOH) showed a single spot andMS showed one single desired peak of 478 (M+H). The mixture wasconcentrated and the excess piperidine was removed by azeotropicconcentration with toluene (three times in 50 mL). The residue wasdissolved in water with addition of MeOH, and then 1N NaOH was addeduntil no further precipitate was formed with adequate stirring. Themixture was concentrated to remove any residual MeOH, filtered andwashed with water. The collected free form solid (16.55 g, 34.7 mmolafter dried under vacuum) was dissolved in warm MeOH (100 mL) andtreated with 70 mL of 1N HCl (70 mmol) to form a di-HCl salt and themixture was concentrated to dryness. The residual solid was crystallizedin MeOH/EtOAc to give a pure product Compound 30 (16.0 g, 82%). ¹H NMR(400 MHz, CD₃OD) δ 8.13 (d, J=8.8 Hz, 2H), 7.58 (d, J=8.8 Hz, 1H), 7.29(d, J=2.3 Hz, 1H), 7.14 (d, J=8.8 Hz, 2H), 7.02 (dd, J=2.3, 8.8 Hz, 1H),4.21 (t, J=5.57 Hz, 2H), 4.19 (t, J=5.57 Hz, 2H), 3.59 (m, 4H), 3.34 (m,4), 2.99 (m, 4H), 2.28 (tt, J=2.3, 5.6 Hz, 4H), 2.00-1.50 (m, 12H); MS(ES⁺) m/z 478.5.

Example 6 Synthesis of Compound 51

Compound 51. Analogs such as Compound 51 may be prepared usingsemi-automated synthesis (SAS) using the following procedure. A mixtureof Compound 40 (27.0 mg, 0.071 mmol) and cyclopentylamine (98.6 mg, 1.16mmol) in N-methylpyrrolidinone (0.5 mL) was heated by microwave at 180°C. for 300 seconds. The crude reaction mixture was purified by LC/MSunder acidic conditions. The collected fractions containing the desiredproduct were checked by analytical LC/MS for purity. The overall purityof this compound was >95% with an m/z=478.5. The two fractionscontaining pure product were combined and evaporated to give productCompound 51 (22.5 mg, 66.4%).

Example 7 Exemplary Compounds

The following analogs in Table 1 below have been prepared. Thesecompounds may be prepared following the procedure for the preparation ofCompound 23 (Example 4) and/or Compound 30 (Example 5) using thecorresponding dichloroalkanes and the side chain amines identified inthe table. These compounds may also be prepared following the procedurefor the preparation of Compound 51 (Example 6).

TABLE 1 Exemplary Compounds Com- pound Structure Side chain amine Com-pound 1

Com- pound 2

Com- pound 3

Com- pound 4

Com- pound 5

Com- pound 6

Com- pound 7

Com- pound 8

Com- pound 9

Com- pound 10

Com- pound 11

Com- pound 12

Com- pound 13

Com- pound 14

Com- pound 15

Com- pound 16

Com- pound 17

Com- pound 18

Com- pound 19

Com- pound 20

Com- pound 21

Com- pound 22

Com- pound 23

Com- pound 24

Com- pound 25

Com- pound 26

Com- pound 27

Com- pound 28

Com- pound 29

Com- pound 30

Com- pound 31

Com- pound 32

Com- pound 33

Com- pound 34

Com- pound 35

Com- pound 36

Com- pound 37

Com- pound 38

NMR Data for Select Compounds in Table Above:

Compound 1: ¹H NMR (400 MHz, CD₃OD) δ 8.14 (d, J=8.8 Hz, 2H), 7.58 (d,J=8.8 Hz, 1H), 7.33 (d, J=2.4 Hz, 1H), 7.16 (d, J=8.8 Hz, 2H), 7.06 (dd,J=2.2, 8.8 Hz, 1H), 4.28 (m, 4H), 3.10 (m, 4H), 2.68 (s, 6H), 2.57 (s,6H).

Compound 2: ¹H NMR (400 MHz, CD₃OD) δ 8.14 (d, J=8.8 Hz, 2H), 7.59 (d,J=8.8 Hz, 1H), 7.30 (d, J=2.4 Hz, 1H), 7.15 (d, J=8.8 Hz, 2H), 7.04 (dd,J=2.4, 8.8 Hz, 1H), 4.21 (m, 4H), 3.34 (m, 4H), 2.93 (s, 6H), 2.92 (s,6H), 2.68 (s, 6H), 2.26 (m, 4H).

Compound 3: ¹H NMR (400 MHz, CD₃OD) δ 8.15 (d, J=8.8 Hz, 2H), 7.55 (d,J=8.8 Hz, 1H), 7.24 (d, J=2.4 Hz, 1H), 7.10 (d, J=8.8 Hz, 2H), 6.98 (dd,J=2.4, 8.8 Hz, 1H), 4.10 (m, 4H), 3.25 (m, 4H), 1.90 (m, 4H), 1.78 (m,4H), 1.59 (m, 4H).

Compound 4: ¹H NMR (400 MHz, CD₃OD) δ 8.18 (dd, J=1.6, 8.0 Hz, 1H), 7.72(d, J=9.0 Hz, 1H), 7.60 (m, 1H), 7.37 (d, J=2.4 Hz, 1H), 7.30 (d, J=8.4Hz, 1H), 7.21 (dd, J=7.2, 8.0 Hz, 1H), 7.09 (dd, J=2.6, 9.0 Hz, 1H) 4.39(t, J=5.6 Hz, 2H), 4.20 (t, J=5.8, 2H), 3.22 (dd, J=7.6, 8.4 Hz, 2H),3.05 (s, 6H), 2.82 (s, 6H), 2.38 (m, 2H), 2.23 (m, 2H).

Compound 5: ¹H NMR (400 MHz, CD₃OD) δ 7.79 (d, J=8.0 Hz, 1H), 7.75 (dd,J=1.6, 2.4 Hz, 1H), 7.63 (d, J=8.8 Hz, 1H), 7.50 (dd, J=8.0, 8.0 Hz,1H), 7.32 (d, J=2.4 Hz, 1H), 7.18 (dd, J=2.6, 8.6 Hz, 1H), 7.06 (dd,J=2.4, 8.8 Hz, 1H) 4.21 (m, 4H), 3.32 (m, 4H), 2.91 (s, 6H), 2.90 (s,6H), 2.25 (m, 4H).

Compound 20: ¹H NMR (400 MHz, CD₃OD) δ 8.10 (d, J=8.8 Hz, 2H), 7.55 (d,J=8.8 Hz, 1H), 7.25 (d, J=2.4 Hz, 1H), 7.10 (d, J=8.8 Hz, 2H), 6.99 (dd,J=2.4, 8.8 Hz, 1H), 4.12 (m, 4H), 3.04 (bd, J=10.8 Hz, 4H), 2.66 (bs,8H), 2.57 (m, 4H), 2.15-1.90 (m, 14H), 1.82 (m, 8H), 1.60 (m, 4H).

Compound 27: ¹H NMR (400 MHz, CD₃OD) δ 8.13 (d, J=9.2 Hz, 2H), 7.58 (d,J=8.8 Hz, 1H), 7.29 (d, J=2.4 Hz, 1H), 7.13 (d, J=9.2 Hz, 2H), 7.03 (dd,J=2.2, 8.6 Hz, 1H), 4.20 (m, 4H), 3.60-3.10 (m, 12H), 2.98 (s, 6H), 2.93(s, 12H), 2.25 (m, 8H).

Compound 35: ¹H NMR (400 MHz, CD₃OD) δ 8.10 (d, J=8.8 Hz, 2H), 7.55 (d,J=8.8 Hz, 1H), 7.25 (d, J=2.4 Hz, 1H), 7.10 (d, J=8.8 Hz, 2H), 6.99 (dd,J=2.4, 8.8 Hz, 1H), 4.10 (m, 4H), 3.60 (m, 8H), 2.65 (m, 8H), 2.35 (m,18H), 2.02 (m, 4H).

Compound 36: ¹H NMR (400 MHz, CD₃OD) δ 8.14 (d, J=8.8 Hz, 2H), 7.55 (d,J=8.8 Hz, 1H), 7.30 (d, J=2.4 Hz, 1H), 7.13 (d, J=8.8 Hz, 2H), 7.03 (dd,J=2.4, 8.8 Hz, 1H), 4.22 (m, 4H), 3.92 (bs, 4H), 3.87 (m, 4H), 3.60-3.20(m, 12H), 3.00 (s, 6H), 2.92 (s, 12H), 2.30 (m, 4H).

Compound 37: ¹H NMR (400 MHz, CD₃OD) δ 8.14 (d, J=8.8 Hz, 2H), 7.59 (d,J=8.8 Hz, 1H), 7.31 (d, J=2.4 Hz, 1H), 7.15 (d, J=8.8 Hz, 2H), 7.04 (dd,J=2.4, 8.8 Hz, 1H), 4.23 (m, 4H), 3.58 (m, 8H), 3.55-3.35 (m, 6H), m3.23 (m, 6H), 2.96 (s, 6H), 2.90 (m, 12H), 2.30 (m, 4H), 2.05 (m, 8H).

Compound 38: ¹H NMR (400 MHz, CD₃OD) δ 8.13 (d, J=8.8 Hz, 2H), 7.58 (d,J=8.8 Hz, 1H), 7.29 (d, J=2.0 Hz, 1H), 7.13 (d, J=8.8 Hz, 2H), 7.02 (dd,J=2.4, 8.8 Hz, 1H), 4.20 (m, 4H), 3.30-3.50 (m, 4H), 3.18 (m, 6H),2.85-2.96 (m, 18H), 2.28 (m, 4H), 1.82 (m, 6H).

Example 8 Synthesis of Compound 34

Compound 34. Referring to the scheme above, a mixture of4-aminoresorsinol hydrochloride (5.3 g, 33 mmol), 4-benzyloxybenzoicacid (5.0 g, 22 mmol), 1-hydroxybenzotriazole (5.9 g, 44 mmol),benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (23g, 44 mmol) and N,N-diisopropylethylamine (19 mL, 110 mmol) indimethylformamide (DMF) (50 mL) was stirred at 0° C. to room temperatureovernight. After concentration to remove DMF, the reaction mixture waspurified by silica gel chromatography (20% to 80% EtOAc/hexane, EtOAcand 30% MeOH/EtOAc) to give desired amide Compound 41 (or/and ester, 7.3g, 95%).

A solution of Compound 41 (7.3 g, 22 mmol) in acetic acid (90 mL) washeated at 120° C. for 3 hours. The reaction mixture was cooled to roomtemperature and concentrated. The residue was purified by silica gelchromatography (10% to 30% EtOAc/hexanes) to give the desired productCompound 42 (2.0 g, 29%) as light yellow solid. ¹H NMR (400 MHz, CD₃OD)δ 8.09 (d, J=9.1 Hz, 2H), 7.47-7.4 (m, 6H), 7.16 (d, J=9.1 Hz, 2H), 7.02(d, J=2.4 Hz, 1H), 7.02 (dd, J=2.4, 8.5 Hz, 1H), 5.18 (s, 2H); MS (ES⁻)m/z 316.4.

A mixture of Compound 42 (1.77 g, 5.58 mmol), 1-bromo-3-chloropropane(1.6 mL, 17 mmol) and potassium carbonate (2.7 g, 20 mmol) in 10 mL DMSOat room temperature was stirred for 2.5 hours until the reaction wascompleted by TLC. The reaction was quenched by addition of water andthen extracted five three times with EtOAc. The combined organic phasewas dried over Na₂SO₄, filtered and concentrated. The residue wascrystallized from ethyl acetate-hexane to give Compound 43 as a lightsolid (1.93 g, 88%).

To a suspension of Compound 43 (1.10 g, 2.8 mmol) in 40 mL of ethanolwas added 5 mL of dimethylamine at −78° C. The mixture was sealed in asealed tube and heated at 100° C. for 10 hours. The mixture wasconcentrated to give Compound 44 (1.1 g, 98%). The product was carriedon to next step reaction.

A mixture of Compound 44 (1.1 g, 2.7 mmol) and palladium hydroxide (7mg) in 32 mL of methanol and 5 mL of dichloromethane was stirred under ahydrogen balloon at room temperature over night. The mixture wasfiltered through celite and the filtrate was concentrated to give crudeproduct Compound 45. Assuming a theoretical yield of 0.85 and theproduct was carried on to the next step directly without furtherpurification.

A mixture of Compound 45 (200 mg, 0.64 mmol), 1-bromo-3-chloropropane(0.32 mL, 3.2 mmol) and potassium carbonate (470 mg, 3.4 mmol) in 4 mLDMSO at room temperature was stirred for 2 hours until the reaction wascompleted by TLC. The reaction was quenched by addition of water andthen extracted four times with EtOAc. The combined organic phase wasdried over Na₂SO₄, filtered and concentrated. The residue was purifiedby preparative TLC (4% Et₃N in 5% MeOH/THF) to give Compound 46 (170 mg,68%).

A mixture of Compound 46 (30 mg, 0.08 mmol),N,N,N′-trimethyl-1,3-propane-diamine (50 mg, 0.43 mmol) in 2 mL ofethanol under a sealed tube was heated at 100° C. for 10 hours. Themixture was concentrated and the residue was purified by HPLC to give 8mg of Compound 34 as a white solid (8 mg, 20%). ¹H NMR (400 MHz, CD₃OD)δ 8.10 (d, J=8.8 Hz, 2H), 7.55 (d, J=8.8 Hz, 1H), 7.25 (d, J=2.3 Hz,1H), 7.10 (d, J=8.8 Hz, 2H), 7.00 (dd, J=2.3, 8.8 Hz, 1H), 4.14 (t,J=5.9 Hz, 2H), 4.10 (t, J=6.2 Hz, 2H), 2.69 (m, 4H), 2.56 (m, 4H), 2.39(s, 12H), 2.36 (s, 3H), 2.04 (m, 4H), 1.77 (m, 2H); MS (ES⁻) m/z+H469.55.

Example 9 In Vitro Biological Activity

A. Primary Assay. The primary assay involved transfecting humanembryonic kidney epithelial (HEK) cells with TLR9. More specifically,HEK293 fibroblast cells (ATCC No. CRP-1573, American Type CultureCollection, Manassas, Va.) were transfected with pcDNA3.1D/V5-His-TOPO®plasmid (Invitrogen, Carlsbad, Calif.) encoding human TLR9 (directlyinserted as a Tag polymerase-amplified PCR product) and fireflyluciferase under the control of three NF-kB binding sites contained inthe E-selectin-1 promoter, which was cloned into a pGL3-Enhancer Vector(Promega Corp, Madison, Wis.). The compounds tested in this assay arelisted in Table 1, below. Each compound was added to cells 30 min.before stimulation with Oligo CpG 2006, a synthetic phosphorothioateoligonucleotide (Hartmann et al., J Immunol. 164:1617-24 (2000)), withnucleotide sequence 5′-TCG TCG TTT TGT CGT TTT GTC GTT-3′ (SEQ ID NO:1).The cells were incubated overnight at 37° C. The luciferase substrateSteady-Glo® (Promega) was added to the wells, and luminescence wasmeasured to determine the extent of TLR9-driven gene activation. Theluminescence data was used to calculate an IC₅₀ for each sample. TheIC₅₀ is defined as the concentration of compound that suppresses theluminescence to 50% of that observed in the absence of compound, inother words, with full stimulation. The results of this assay are shownin Table 2, below. The IC₅₀ of the Comparator was found to be 0.16micromolar. The IC₅₀ for Compound 1 was found to be 0.06 micromolar andthe IC₅₀ for the remaining compounds was less than 0.04 micromolar.

B. Secondary Assays.

(1) HEK/TLR7Activation Assay: The same compounds were tested in the sameway as the primary assay with the following differences. The plasmidused to transfect the HEK cell line encoded for human TLR7 instead ofTLR9, and R848 was used to stimulate the cells instead of Oligo CpG2006. The results of this assay are also shown in Table 2. For eachcompound tested the IC₅₀ from the HEK/TLR9 activation assay, above, wassignificantly lower than the IC₅₀ from the HEK/TLR7 assay, indicating agreater specificity for TLR9 than for TLR7.

(2) Splenocyte CpG 1668 Assay: Some of the compounds tested in theprimary assay were also assayed for suppression of mouse spleen IL-6production in response to stimulation with Oligo CpG 1668, a syntheticphosphorothioate oligonucleotide (Krieg et al. Nature. 374:546-9(1995)), with nucleotide sequence 5′-TCC ATG ACG TTC CTG ATG CT-3′ (SEQID NO:2). Each compound was added to dissociated splenocytes beforeaddition of stimulatory oligonucleotide. Cells were stimulated inculture for 72 hrs and the supernatant was removed for ELISA analysis ofIL-6 level. Here the IC₅₀ is defined as the concentration of compoundthat suppresses cytokine production to 50% of that observed in theabsence of compound, i.e., full stimulation. All compounds that weretested, except the comparator, inhibited stimulation of mousesplenocytes with an IC₅₀ below 1 μM. The results of this assay are shownin Table 2, below.

(3) Spleen CT Glo Assay: To determine if compounds had adverse effectson living cells, dissociated mouse splenocytes were incubated with someof the compounds for 72 hours. At the end of the incubationCelliter-Glo™ reagent (Promega Corporation, Madison, Wis.) was added andluminescence was read per manufacturer's instructions. This reagentmeasures ATP as an indication of cell metabolism. Dead cells will notproduce signal. IC₅₀ is defined as the concentration of compound thatsuppresses viability to 50% of that observed in the absence of compound.The few compounds that measurably affected viability did so atconcentrations far above those that affected cytokine release inresponse to TLR9 stimulation. The results are shown in Table 2. Theseresults indicate that the TLR9 suppression was not due to cell death orweakness.

(4) PBMC CpG 2216 Assay: A subset of compounds tested as described abovewere also assayed for suppression of human peripheral blood mononuclearcells (PBMC) α-interferon response to stimulation with Oligo CpG 2216, asynthetic oligo phosphorothioate/phosphodiester oligonucleotide (Vollmeret al. Eur J. Immunol. 34:251-62 (2004)), with nucleotide sequence5′-ggG GGA CGA TCG TCg ggg gG-3′ (small letters indicatephosphorothioate linkages, all others are phosphodiester) (SEQ ID NO:3).Each compound was added to Ficoll-Paque™ (GE Healthcare, UK) to separatemononuclear cells from healthy volunteer donors before addition ofstimulatory oligonucleotide. Cells were stimulated in culture for 72 hrsand the supernatant was removed for ELISA analysis of α-interferon levelwas performed using a VeriKine™ Human IFN-Alpha ELISA kit (PestkaBiomedical Laboratories, N.J.). All the compounds tested in this assayinhibited stimulation of human PBMC with an IC₅₀ below 1 μM.

Table 2 below shows the compounds tested and results obtained from eachof the assays described and discussed above.

TABLE 2 In Vitro Assay Results HEK/ Spleen HEK/ TLR7 SplenocyteCellTiter- PBMC Compound TLR9 IC₅₀ CpG 1668 Glo CpG2216 Number IC₅₀ (μM)(μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) Comparator 0.160 2.69 1.02 Compound 10.031 7.80 0.29 Compound 2 0.0154 2.21 0.048 >10 0.5 Compound 3 0.02301.4 Compound 4 0.0120 1.8 Compound 5 0.0110 2.0 Compound 6 0.0202 8.41Compound 7 0.0233 1.31 >10 Compound 8 0.0197 1.23 7.82 Compound 9 0.129.05 Compound 10 0.0153 0.35 0.026 9.8 Compound 11 0.0048 0.47 0.002 100.6 Compound 12 0.0058 0.78 0.004 >10 0.5 Compound 13 0.0071 0.46 0.0037.07 Compound 14 0.0056 0.62 0.004 >10 Compound 15 0.0232 2.24 >10Compound 16 0.0055 0.43 0.008 >10 0.4 Compound 17 0.0053 0.17 0.006 >10Compound 18 0.0192 1.40 9.9 Compound 19 0.0276 1.10 10 Compound 200.0125 1.82 0.006 >10 0.7 Compound 21 0.0133 1.24 9.58 Compound 220.0087 0.50 0.010 7.11 Compound 23 0.0111 1.55 0.018 >10 0.6 Compound 240.0064 0.60 0.008 >10 Compound 25 0.0182 0.90 0.048 >10 Compound 260.0124 0.99 0.042 >10 Compound 27 0.0034 0.98 0.004 8.96 Compound 280.0125 1.55 >10 Compound 29 0.0193 0.78 0.042 10.39 Compound 30 0.01211.14 0.025 >10 0.5 Compound 31 0.0072 0.44 0.006 >10 0.2 Compound 320.0096 0.58 0.010 >10 Compound 33 0.100 8.52 Compound 34 0.004 0.8Compound 35 0.029 1.4 0.04 Compound 36 0.038 >2.5 Compound 37 0.030 >2.5Compound 38 0.034 >2.5

Example 10 In Vivo Biological Activity: Short Term CpG Stimulation

Short Term CpG Stimulation Assay: Compounds identified as having potencyin the HEK/TLR9 and splenocyte CpG 1668 assays described in Example 9,above, were tested in an in vivo short term CpG stimulation assay. Micewere orally administered compound in water, and challenged within 90minutes by subcutaneous (s.c.) injection of 30 micrograms of oligo CpG1668. Two hours later serum was taken and assayed for IL-6 using ELISAanalysis. The results of this assay are shown in Table 3, below. Resultsfrom assays repeated on different dates are set forth as multiplenumbers separated by commas.

TABLE 3 CpG Stimulation Assay Compound Percent short-term number IL-6suppression Comparator  7, 32 Compound 13 40, 44 Compound 14  0 Compound16 50, 45 Compound 20  0 Compound 22 36 Compound 23 91, 75 Compound 2681, 69 Compound 27 30 Compound 30 93, 79 Compound 34 26 Compound 35 38

Table 3 shows that at least 69% suppression of IL-6 was observed 1.5hours after oral administration of 20 mg/kg Compound 23, Compound 30, orCompound 26. At least 75% suppression of IL-6 was observed after oraladministration of Compound 23 and Compound 30. No suppression wasobserved after administration of two of the compounds tested, Compound14 and Compound 20, despite the high levels of suppression exhibited inthe in vitro assays described above. The remaining compounds exhibitedvarying levels of IL-6 suppression.

Example 11 In Vivo Biological Activity: Lymph Node Response

Two compounds assayed as described in Example 10 above, Compound 23 andCompound 30, showed a particularly high level of suppression. Mice wereorally administered one of those two compounds or hydroxychloroquine(“HCQ”) at either 20 mg/kg or 60 mg/kg daily for two weeks. After invivo dosing, lymph nodes were removed and stimulated in vitro with CpG1668 at 1 μg/ml for 72 hrs, after which supernatants were removed andassayed by ELISA for IL-6. Results of this assay are shown in FIG. 1.Levels of dosing are indicated after each compound name appearing in theFigure.

The results in FIG. 1 show that both Compound 2 and Compound 23suppressed IL-6 release in the lymph nodes, and Compound 23 showedparticularly high levels of suppression even upon administration of only20 mg/kg.

Example 12 In Vivo Biological Activity: Spontaneous Lupus Models

A. MRL/MpJ-faslpr/J Experiment 1.

In this experiment, MRL/MpJ-faslpr/J mice (“MRL/lpr”) obtained fromJackson Laboratories were dosed orally with Compound 23 at 20 or 60mg/kg in water. MRL/lpr mice have a defect in lymphocyte death andspontaneously develop autoantibodies including the anti-nuclearantibodies found in human systemic lupus erythematosus (SLE). Dosing wasperformed daily five days a week beginning at 5 weeks of age. Blood andurine samples were taken prior to dosing on day one and every three tofour weeks thereafter. An ELISA assay was used to measure serumanti-dsDNA before and after 7 weeks of dosing. The assay was conductedusing a Mouse Anti-dsDNA Total Ig Qualitative ELISA kit (AlphaDiagnostic International, San Antonio. TX). The results are shown inFIG. 2. FIG. 2 shows that there was a dose-titrating effect on thisautoantibody, with statistically significant suppression of the meanOptical Density at 450 nm (OD₄₅₀) versus vehicle-dosed animals. Thestatistical difference was still observed at 10 weeks of dosing, butthen compound-treated groups became more similar to vehicle-treatedanimals dosed with dH₂O, given orally, at 12 weeks and beyond. Noevidence of dosage-related toxicity was seen as judged by generalappearance or body weight measurements.

Anti-nuclear antibodies (“ANA”) in this experiment were also examined byexposing fixed HepG2 cells, obtained as fixed samples on slides fromAntibodies Inc., to diluted serum from the mice dosed with Compound 23at 20 or 60 mg/kg, as described above, and staining for mouseimmunoglobulin using the kit reagents supplied by Antibodies Inc.according to the kit instructions. Intensity of fluorescent staining wasthen read by microscope, and scores were assigned values between − and+++. Sample identifications were blinded for data collection.

FIG. 3 is a graphic illustration of the results of the ANA testing. FIG.3 shows that there was a dose-dependent suppression of ANA development,in that vehicle-treated mice gradually became ANA positive between 5 and12 weeks of age, while sera from compound treated mice showed muchslower progression to positive ANA staining.

B. MRL/lpr Experiment 2.

In this SLE experiment, dosing, groups were as described in Table 4below. Two dose levels of Compound 23 were compared to HCQ andcyclophosphamide (Cytoxan®). HCQ and cyclophosphamide are currently usedin the treatment of cutaneous and systemic lupus, respectively.Cyclophosphamide showed consistent suppression of serum anti-dsDNAlevels. This difference was statistically significant at the 8 and 12week dosing points, when tested vs. vehicle by ANOVA. Results from ELISAassays of optical densities at 450 nanometers (OD₄₅₀) for individualanimal sera pre- and post-dosing for 12 weeks are shown in FIG. 4. Serawere assayed at a 1:600 dilution in PBS. (Mouse Anti-dsDNA Total IgQualitative ELISA kits, Alpha Diagnostic International). Dosing wasconducted by either oral (p.o.) or intraperitoneal (i.p.)administration. Individual data points from this assay are illustratedin FIG. 4. FIG. 4 shows that at 12 weeks, there was also a statisticallysignificant difference in mean dsDNA level between vehicle and the highdose of Compound 23.

No effect on proteinuria (measured by BCA protein assay) or skin lesionswas seen by any compound in this experiment.

TABLE 4 Dosing Regimen Dose Route of Group Compound (mg/kg) admin.Regimen 1 Compound 23 60 p.o. M-F 2 Compound 23 20 p.o. M-F 3Hydroxychloroquine 60 p.o. M-F 4 Vehicle — p.o. M-F 5 Cyclophosphamide50 i.p. Every 10 days

Example 13 In Vivo Biological Activity: NZBWF1/J Spontaneous Lupus Model

An in vivo study of Compound 23 and Compound 30 was conducted in anNZBWF1/J mouse model. Mice were obtained from Jackson Laboratories,divided into groups of 12, and dosed orally with compounds in water fivedays a week beginning at 5 months of age. Dosing details are provided inTable 5 below:

TABLE 5 Dosing Regimen Dose Route of Dose/head Group Compound (mg/kg)admin. regimen (ml) 1 Compound 23 60 p.o. M-F 0.2 2 Compound 23 20 p.o.M-F 0.2 3 Compound 30 60 p.o. M-F 0.2 4 Compound 30 20 p.o. M-F 0.2 5Vehicle (dH₂O) — p.o. M-F 0.2

Neither of the compounds tested in this study, Compound 23 or Compound30, showed an effect on anti-dsDNA during the 16 weeks of treatmentafter dosing with 20 mg/kg or 60 mg/kg of compound. Results of the studyare illustrated in FIG. 5. Compound 30, which showed a somewhat higherpotency than Compound 23 in the short-term assay, gave some suppressionof anti-dsDNA at the high dose of 60 mg/kg, but, this was notstatistically significant. ANA was measured in the same experiment, andalthough only 5 of 12 vehicle-treated animals progressed to scores of+/− or higher there was no effect of compound on distribution of ANAscores after 12 weeks of treatment. Results of the ANA scoring areillustrated in FIG. 6.

Compound 23 and Compound 30 consistently produced a dose-related weightloss in the NZBWF1/J mouse strain after 3 to 4 months of dosing.Histological examination of muscle, brain, kidney and spleen showed noobvious pathology in hematoxylin and eosin staining.

Daily oral dosing with 60 and 20 mg/kg Compound 23 resulted insuppression of anti-dsDNA in the MLR/lpr model of SLE. Compound 23 alsoshowed a dose-dependent inhibition of anti-nuclear antibody development.Cyclophosphamide suppressed anti-dsDNA in the MRL/lpr model, whilehydroxychloroquine had no detectable effect. Further, the test ofCompound 23 in the NZB/W system showed no effect on anti-dsDNA, ANA, orproteinuria. Dose-dependent weight loss was observed after severalmonths of dosing in NZB/W mice.

Example 14 In Vivo Biological Activity: Experimental AutoimmuneEncephalitis Multiple Sclerosis Model

Experimental autoimmune encephalitis (EAE) is an induced autoimmunity inmice that mimics human multiple sclerosis, including autoantibody andcellular responses targeted against central nervous system antigens. Toinduce EAE, groups of 8 male C57BL/6J mice (Jackson Laboratories) wereimmunized subcutaneously on day 0 with the synthetic peptide MOGp35-55(Becher et al. J. Clin. Invest. 112:1186-1191 (2003)), with amino acidsequence MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO:4) at a dose of 200 μg/mousein complete Freund's adjuvant (CFA). At the same time the animalsreceived pertussis toxin, i.p. at 30 ng/mouse. On day 2 the pertussisinjection was repeated. On day 7 the mice were boosted with MOGp35-55,200 μg/mouse in incomplete Freund's. Beginning on day 9 the mice weredosed daily by oral gavage with Compound 23 in water at 20 mg/kg or 60mg/kg. Symptoms were scored by observing degree and location ofparalysis of the tail and limbs, the average score for each group of 8mice is shown.

The results of this study are shown in FIG. 7. It can be observed thatMOG-induced EAE was suppressed significantly in mice administered 20mg/kg Compound 23, compared to the control mice, and was suppressedfurther in mice administered 60 mg/kg of the same compound.

Example 15 In Vivo Biological Activity: Collagen Induced Arthritis

The type II collagen induced arthritis model was used for the purpose ofin vivo evaluation of Compound 2 for the treatment of arthritis. In thismodel, arthritis was induced in male DBA/1J (Jackson Labs) mice usingcomplete Freund's adjuvant. A first priming/immunization dose of 300 μgof bovine type II collagen emulsified in an equal volume of completeFreund's adjuvant is injected subcutaneously (s.c.) at least 5 mm fromthe base of the tail.

Starting on day 9 after immunization and each day for 5 days per weekthereafter, each mouse was dosed orally with 6 mg/kg, 20 mg/kg, or 60mg/kg of Compound 2 or placebo 5 days per week.

A second immunization dose was administered 21 days after the firstimmunization by s.c. injection above the first immunization site of 200μg of bovine type II collagen emulsified in an equal volume ofincomplete Freund's adjuvant. To further promote arthritis progression,animals receive an intraperitoneal injection of lipopolysaccharide (LPS)(1 μg/mouse in PBS) three days after the second immunization.

At 21, 25, 28, and 31 days after the first immunization dose, theanimals were examined for evidence of paw swelling and assigned anarthritis score at each time point. The results of this assay are shownin FIG. 8. Suppression of paw swelling was observed after dosing with 6and 20 mg/kg, but, the results after dosing with 60 mg/kg were notclear. The anomalous 60 mg/kg result suggest the need for furtherstudies to determine an appropriate upper dosing level for thisparticular animal model.

Example 16 In Vivo Biological Activity: Cecal Ligation and PunctureSurgery

The cecal ligation and puncture (CLP) model involves a surgicalprocedure where an abdominal incision is made to expose the cecum (aneasily isolated section of the digestive tract) and a portion of thececum is ligated. The cecum is then punctured and a small amount of theintestinal contents is extruded. The incision is closed and the animalsare given resuscitative saline and antibiotics. Sepsis developsprimarily due to bacteria from the extruded intestinal contents anddisease development occurs with a rapidity and severity that depends onthe size and number of punctures in the cecum.

A. Cecal Ligation and Puncture Surgery.

Anesthesia is induced in mice by i.p. administration of a combination ofketamine, xylazine and acepromazine. The animal's abdominal region canbe closely shaved and a transponder for measuring body temperature isimplanted subcutaneously. The procedure takes place in an area suitablefor aseptic survival surgery. Animals are placed on a warming, padthroughout the procedure. The abdominal area is prepped with a minimumof three alternating wipes of povidone/iodine and alcohol.

A midline incision is made to open the abdomen and expose the cecum. Thececum is removed from the abdominal cavity using cotton-tippedapplicators moistened with saline. The tip of the cecum is ligated using4-0 sutures just distal to the ileocecal valve. Cecal contents areshifted to one end and the cecum is then punctured with a sterileneedle. Pressure is applied to extrude a small amount of material fromthe cecum into the peritoneal cavity. The ligature is left in place andnot removed. The muscle and fascia is closed with continuous 4-0 suturesand the skin is closed with wound staples.

Immediately after surgery, 5 ml per 100 g body weight of warmresuscitative saline is injected s.c. Baseline readings for temperatureand body weight are recorded. One hour after surgery, a 20 mg/kg dose ofthe antibiotic moxifloxacin is administered i.p. along with anyanti-sepsis drug candidates being tested. Antibiotics are administeredat 24 hr intervals for the duration of the study. The dose of antibioticadministered is not intended to eradicate the bacteria released by CLP,but is instead used to model the clinical scenario where humans would bereceiving antibiotics although they may be ineffective. Althoughmoxifloxacin is associated with prolongation of the QT interval, it isconsidered safe for clinical use as long as unfavorable drug,interactions are avoided (Torres et al., J Surg Res. 125: 88-93 (2005))and the antibiotic has previously proven safe and effective in anothermouse sepsis model (Alkorta et al., Int J. Antimicrob. Agents 25 (2):163-7, (2005)).

B. Administration of Test Compound.

Compounds are administered approximately 1 hr after surgery at the timeof antibiotic injection, although the timing can vary. Compounds areadministered twice per day depending on the pharmacokinetic propertiesof the candidate compounds. Administration of test compound is performedby any of the following routes according to established guidelines foradministration: oral, intraperitoneal, subcutaneous, or intravenousinjection through the tail vein.

Groups of around 10 animals are used. Drug evaluation experiments have agroup that experiences cecal ligation and puncture and receives onlyvehicle (control group), and a group(s) that experiences CLP andreceives a test treatment compound. Compounds are administered 1 hrpost-surgery and animals are monitored for survival every two hoursduring the day. Body temperatures are taken via transponder every twohours and animal health is monitored at each point. Body weights aretaken at 4 hr intervals. Animals are monitored for a period of at least8 hrs each day. Serum samples are taken for measurement of cytokinelevels. Mortality is monitored, and used as a measure of the efficacy ofdrug treatment.

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein one of R⁵, R⁶, orR⁷ is a group of formula (a):

and when R⁵ is (a), R⁶ and R⁷ are both H; when R⁶ is (a), R⁵ and R⁷ areboth H; and when R⁷ is (a), R⁵ and R⁶ are both H; i and j are the sameand are 0, 1, 2, 3, or 4; R¹ and R⁴ are the same and selected from thegroup consisting of H, CH₃, and CH₂CH₃; R² is CH₃ and R³ is selectedfrom the group consisting of (CH₂)_(h)N(CH₃)₂ and(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(CH₃)₂, wherein h is 2, 3, or 4; or R² and R³are the same and selected from the group consisting of: (CH₂)_(k)CH₃,wherein k is 0, 1 or 2, (CH₂)_(m)N(CH₂CH₃)₂, wherein m is 2 or 3,(CH₂)_(n)N(CH₃)₂, wherein n is 2, 3 or 4, (CH₂)_(p)O(CH₂)_(q)N(CH₃)₂,wherein p and q are the same and are 2 or 3; a group of formula (b):

 wherein u is 0 or 1; or a group of formula (c):

 wherein v is 0 or 1, Z is N or CH, and X is O or NCH₃; or R¹—N—R² andR³—N—R⁴ are the same and selected from the group consisting of: a groupof formula (d):

 and a group of formula (e):

wherein r is 1, 2, or 3, Y is CH or N, R⁸ is H, CH₃, CH(CH₃)₂, N(CH₃)₂,CH₂OCH₃, or a group of formula (f):

 wherein t is 0 or 1, and R⁹ is H, CH₂OCH₃, or a group of formula (f).2. The compound of claim 1, wherein i and j are both
 1. 3. The compoundof claim 1, wherein R² and R³ are the same and selected from the groupconsisting of: (CH₂)_(k)CH₃, wherein k is 0, 1 or 2;(CH₂)_(m)N(CH₂CH₃)₂, wherein m is 2 or 3; the group of formula (b); andthe group of formula (c).
 4. The compound of claim 1, wherein R¹—N—R²and R³—N—R⁴ are the same and selected from the group consisting of: agroup of formula (g): a group of formula (h):

a group of formula (i): a group of formula (j):

a group of formula (k): a group of formula (m):


5. The compound of claim 1, wherein: i and j are both 1; R¹ and R⁴ arethe same and selected from the group consisting of H and CH₃; R² is CH₃and R³ is selected from the group consisting of (CH₂)₂N(CH₃)₂ and(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(CH₃)₂; or R² and R³ are the same and selectedfrom the group consisting of: a) (CH₂)_(k)CH₃, wherein k is 0, 1 or 2;b) (CH₂)_(m)N(CH₂CH₃)₂, wherein m is 2 or 3; c) the group of formula(b); and d) the group of formula (c); or R¹—N—R² and R³—N—R⁴ are thesame and selected from the group consisting of: the group of formula(g), the group of formula (h), the group of formula (i), the group offormula (j), the group of formula (k), and the group of formula (m). 6.The compound of claim 1, wherein: R⁵ is the group of formula (a), and R⁶and R⁷ are each H.
 7. The compound of claim 1, wherein the compound isselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim1, wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 9. The compound of claim8, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition comprising a compound of formula (I):

or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, wherein one of R⁵, R⁶, or R⁷ is a group of formula(a):

and when R⁵ is (a), R⁶ and R⁷ are both H; when R⁶ is (a), R⁵ and R⁷ areboth H; and when R⁷ is (a), R⁵ and R⁶ are both H; i and j are the sameand are 0, 1, 2, 3, or 4; R¹ and R⁴ are the same and selected from thegroup consisting of H, CH₃, and CH₂CH₃; R² is CH₃ and R³ is selectedfrom the group consisting of (CH₂)_(h)N(CH₃)₂ and(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(CH₃)₂, wherein h is 2, 3, or 4; or R² and R³are the same and selected from the group consisting of: (CH₂)_(k)CH₃,wherein k is 0, 1 or 2, (CH₂)_(m)N(CH₂CH₃)₂, wherein m is 2 or 3,(CH₂)_(n)N(CH₃)₂, wherein n is 2, 3 or 4, (CH₂)_(p)O(CH₂)_(q)N(CH₃)₂,wherein p and q are the same and are 2 or 3; a group of formula (b):

 wherein u is 0 or 1; or a group of formula (c):

 wherein v is 0 or 1, Z is N or CH, and X is O or NCH₃; or R¹—N—R² andR³—N—R⁴ are the same and selected from the group consisting of: a groupof formula (d):

 and a group of formula (e):

wherein r is 1, 2, or 3, Y is CH or N, R⁸ is H, CH₃, CH(CH₃)₂, N(CH₃)₂,CH₂OCH₃, or a group of formula (f):

 wherein t is 0 or 1, and R⁹ is H, CH₂OCH₃, or a group of formula (f).11. The pharmaceutical composition of claim 10, wherein the compound isselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 12. The pharmaceuticalcomposition of claim 10, wherein the compound is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.
 13. The pharmaceuticalcomposition of claim 10, wherein the compound has the following formula:

or a pharmaceutically acceptable salt thereof.
 14. A method ofpreventing or treating an immunological disorder selected from the groupconsisting of sepsis, lupus, rheumatoid arthritis and multiple sclerosisin a subject, comprising administering to the subject an effectiveamount of a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 15. The method of claim14, wherein the subject is human.
 16. The method of claim 14, whereinthe compound is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 17. The method of claim14, wherein the compound is

or a pharmaceutically acceptable salt thereof.
 18. The method of claim14, wherein the immunological disorder is sepsis.
 19. The method ofclaim 14, wherein the immunological disorder is selected from the groupconsisting of lupus, rheumatoid arthritis and multiple sclerosis. 20.The method of claim 19, wherein the immunological disorder is lupus.21-23. (canceled)